Abstract:

The present invention relates to diagnostic tests, methods and kits that
are useful to assess a subject's risk of developing a pathologic
condition related in part to the presence of HDL oxidation product.
Measuring the quantity of one or more HDL oxidation products present in
the blood is useful in evaluating risk for developing or evaluating the
severity of a disease or evaluating response to treatment for such a
disease as, for instance, cardiovascular disease.

Claims:

1-25. (canceled)

26. A method for assessing efficacy of a therapy for treating a disease
comprising the steps of:A) obtaining a biological sample from a
subject;B) measuring the amount of an high-density lipoprotein ("HDL")
oxidation product in the biological sample;C) obtaining a second
biological sample from the subject at a later time; andD) comparing the
amount of the HDL oxidation product in the biological sample with the
amount of said HDL oxidation product present in the second biological
sample.

45. The method of claim 26, wherein said biological sample is plasma or
urine.

46. The method of claim 26, wherein measuring said HDL oxidation product
is performed by immunoassay or flow cytometry.

47. The method of claim 46, wherein said measuring is performed by a
method selected from the group consisting of an enzyme-linked
immunosorbent assay (ELISA), a lateral flow assay, a fluorescent
polarization assay, a time-resolved fluorescence assay, a microparticle
capture assay, a capillary electrophoresis assay, HPLC and a fluorescence
immunoassay.

48. The method of claim 28, wherein said subject is undergoing treatment
with an agent useful in the treatment of cardiovascular disease.

Description:

FIELD OF THE INVENTION

[0001]The invention relates to diagnostic methods for assessing the risk
of a subject for development of a pathological condition associated with
high levels of oxidative stress induced compounds, in particular,
cardiovascular disease. In addition, methods are described for monitoring
the effectiveness of therapy in a subject, and for establishing a
prognosis in a subject undergoing treatment for a condition such as a
cardiac condition using specific markers of oxidative stress as
indicators of disease progression or inhibition thereof.

[0003]Cardiovascular disease remains the number one killer of people in
the United States today. The diagnosis of CVD is made by assessing a
patient's clinical symptoms, by running laboratory tests to determine
levels of certain enzymes, as well as by coronary angiography,
electrocardiogram, and an exercise stress test (treadmill).

[0004]There are many risk factors that may contribute to the development
of CVD. Certain of these risk factors are modifiable. These include
cigarette smoking, high LDL cholesterol, low HDL cholesterol, diabetes,
hypertension, and physical inactivity. Other contributing risk factors
include obesity, diet, and alcohol consumption. Some risk factors are not
capable of being modified and these include age, sex, race, and family
history.

[0005]The optimal treatment for CVD is prevention and modification of risk
factors. If the disease has progressed beyond prevention and
modification, surgical intervention including percutaneous transluminal
coronary angioplasty (PTCA), coronary bypass, and coronary stents may be
performed and or implanted.

[0006]While the risk factors for CVD are used by physicians in risk
prediction matrices in an attempt to target those individuals who are at
highest risk for development of CVD, thereby allowing these individuals
to modify their lifestyle to lower their risk profile to the extent
possible, these algorithms are still limited in their predictability.
Accordingly, there is a need for expanding these algorithms to take into
account other factors that should be included in a patient's risk profile
for development of CVD.

[0007]It is generally recognized that many disease processes are
associated with the presence of elevated levels of oxidative stress
induced compounds, such as free radicals and reactive oxygen species
(ROS) and reactive nitrogen species (RNS). These include superoxide,
hydrogen peroxide, singlet oxygen, peroxynitrite, hydroxyl radicals,
hypochlorous acid (and other hypohalous acids) and nitric oxide.

[0008]For example, in the eye, cataract, macular degeneration and
degenerative retinal damage are attributed to ROS. Other organs and their
ROS-related diseases include: lung cancer induced by tobacco combustion
products and asbestos; accelerated aging and its manifestations,
including skin damage and scleroderma; atherosclerosis; ischemia and
reperfusion injury, diseases of the nervous system such as Parkinson
disease, Alzheimer disease, muscular dystrophy, multiple sclerosis; lung
diseases including emphysema and bronchopulmonary dysphasia; iron
overload diseases such as hemochromatosis and thalassemia; pancreatitis;
diabetes; renal diseases including autoimmune nephrotic syndrome and
heavy metal-induced nephrotoxicity; and radiation injuries. Diseases of
aging and chronic emotional stress also appear to be associated with a
drop in glutathione levels, which allows ROS to remain active.

[0009]However, while there has been an association of these disease states
with high levels of oxidative stress induced compounds, the reliance of
these compounds for use as a marker of risk for development of these
diseases has not been demonstrated. On the other hand, there is current
evidence in animal studies that oxidation of LDL occurs in vivo, and the
results suggest that this may lead to the formation and build up of
atherosclerotic plaques.

[0017]There is still a need for diagnostic tests to aid in the
characterization of subjects at risk for developing diseases
characterized in part by high levels of oxidative stress-induced
compounds such as HDL oxidation products, in particular, cardiovascular
disease. Furthermore, there is a need to establish whether a specific
therapy is having the appropriate effect in individuals suffering from
such conditions. Thus, prognostic markers or indicators to monitor the
effects of such therapy are also needed.

SUMMARY OF THE INVENTION

[0018]In its broadest aspect, the invention relates to methods and kits
for assessing a pathological condition associated in part with abnormal
levels of HDL oxidation products. In a more particular aspect, the
present invention provides a means for determining whether a subject is
at risk for developing cardiovascular disease or for assessing a
subject's risk of having progressive cardiovascular disease as may be
manifested, for instance by clinical sequelae, including myocardial
infarction, stroke, and peripheral vascular disease, renal disease, or
renal failure. In addition, the invention provides methods for evaluating
the effectiveness of therapy with an agent useful in preventing or
treating cardiovascular disease and for establishing a prognosis in a
patient suffering from a cardiovascular condition, during or after
treatment with agents effective in treating such conditions. The present
invention takes advantage of the discovery that patients having coronary
artery disease have significantly greater levels of oxidized high density
lipoprotein (HDL) products than patients without coronary artery disease.
In particular, the invention provides for measuring such oxidized high
density lipoprotein (HDL) products as a means of assessing a pathological
condition such as a cardiovascular disease.

[0019]Accordingly, a first aspect of the invention provides a method for
assessing a pathological condition in which abnormal levels of oxidized
high density lipoprotein (HDL) products are associated with the
pathological condition. Such assessing may include diagnosing the
pathological condition, determining the risk for developing the
pathological condition, determining the severity of the pathological
condition or monitoring the efficacy of a therapy for the pathological
condition.

[0020]In a second aspect, the invention provides a method for assessing a
subject's risk for developing a cardiovascular disease. An individual who
demonstrates an increase in oxidized high density lipoprotein (HDL)
products, as compared to a predetermined normal reference range, is at
greater risk for developing cardiovascular disease or for having
cardiovascular disease progress as may be evidenced for instance by a
heart attack, stroke, peripheral vascular disease or renal disease than
individuals whose oxidized high density lipoprotein (HDL) product levels
are within a normal reference range. The invention contemplates a risk
matrix whereby correlating an individual's measured oxidized high density
lipoprotein (HDL) product levels with the matrix may be used to predict
the individual's risk for developing or having cardiovascular disease, or
for having progressive cardiovascular disease as may be manifested, for
instance by heart attack.

[0021]A third aspect of the invention provides a method for assessing
efficacy of a therapy useful for treating cardiovascular disease. The
method comprises collecting a series of biological samples from a subject
suffering from cardiovascular disease, the samples may be obtained before
initiation of therapy and/or at one or more times during administration
of therapy. The level of oxidized high density lipoprotein (HDL) products
is quantified using the methods as described herein. Oxidized high
density lipoprotein (HDL) products and a normalization of oxidized high
density lipoprotein (HDL) products correlates with effectiveness of
therapy.

[0022]A fourth aspect of the invention provides a method for monitoring
cardiovascular function in a patient, or for establishing a prognosis in
a patient suffering from a cardiovascular condition using the diagnostic
tests and methods described herein. In addition to establishing the
quantity of oxidized high density lipoprotein (HDL) products, the levels
of such products may be compared to at least one cardiac function test,
either concurrently or at a different time. The values of oxidized high
density lipoprotein (HDL) products may be correlated to a favorable
cardiac function test or to an unfavorable cardiac function test.

[0023]A fifth aspect of the invention provides a method for monitoring
oxidative stress. Oxidative stress has been implicated in the
pathogenesis of diseases including atherosclerosis, acute lung injury,
arthritis, and carcinogenesis as well as the aging process itself. Prior
to the present invention there were no well accepted markers of oxidative
stress in humans, nor has it been established that proposed
"antioxidants" lower or prevent oxidative stress in human disease or
aging. The values of oxidized HDL may be associated with the overall
level of oxidative stress. Acute or chronic forms of oxidative stress in
disorders like acute lung injury or rheumatoid arthritis may result in
increased levels of oxidized HDL. Moreover, the ability of compounds with
proposed antioxidant activities such as vitamin E to actually lower
oxidative stress in humans may be associated with the levels of oxidized
HDL.

[0024]In a particular embodiment, the methods according to the invention
comprise the following steps: [0025]a) obtaining a biological sample from
an individual; [0026]b) measuring the level of one or more oxidized high
density lipoprotein (HDL) products in the biological sample; [0027]c)
comparing the level of one or more oxidized high density lipoprotein
(HDL) products with a range of predetermined values for oxidized high
density lipoprotein (HDL) products wherein the level of one or more
oxidized high density lipoprotein (HDL) products correlates with the
presence of one or more risk factors for the pathological condition.

[0028]Particularly, an increase in the level of one or more oxidized high
density lipoprotein (HDL) products to a value above the normal range
correlates with the presence of, or the pending onset of a pathological
condition. The biological sample may be whole blood or a derivative
thereof, including but not limited to, whole blood cells, whole blood
cell lysates, erythrocytes, plasma, serum, white blood cells, including
leukocytes, neutrophils and monocytes. In other embodiments, the
biological sample may be other tissues or fluids, including but not
limited to cerebral spinal fluid (for neurological diseases),
bronchoalevolar lavage fluid (for lung disease), joint fluid (for
arthritis), and urine (for systemic disorders and disorders of the
kidney, ureters and bladder). In yet other embodiments, the biological
sample may be a specific component of HDL itself, including but not
limited to apolipoprotein apo A-I, apo A-II, apo A-V, apo CI, CII or
CIII, SAA, paraoxonase, platelet activating factor hydrolase (PAF), or
lipids or vitamins associated with HDL. In preferred embodiments, the
pathological condition is cardiovascular disease. Cardiovascular disease
includes, but is not limited to, atherosclerosis, coronary heart disease,
ischemic heart disease, myocardial infarction, angina pectoris,
peripheral vascular disease, cerebrovascular disease, stroke, renal
disease, and other conditions related to or resulting from an ischemic
event.

[0029]The present invention encompasses a risk matrix that may be
developed correlating values of oxidized high density lipoprotein (HDL)
products with risk for developing or progressing or for the severity of
cardiovascular disease or other disorders associated with oxidative
stress and sequelae of the same.

[0030]The oxidized high density lipoprotein (HDL) products that are
quantified may be any oxidation product indicative of cell injury such as
those that react with peroxynitrite or hypochlorous acid. These oxidized
products are the product of oxidation of one or more amino acids such as
tyrosine, of the lipid portions of the HDL or of molecules in conjuction
with the HDL complex such as a vitamin. Preferred oxidized high density
lipoprotein (HDL) products may include a product of apo A1 and may be
selected from the group consisting of 3-nitrotyrosine,
3,5-dinitrotyrosine, 3-chlorotyrosine, nitrophenyl alanine, chlorophenyl
alanine, o',o'-dityrosine, ortho-tyrosine, meta-tyrosine, WG-4
(cross-linked tryptophan-glycine), oxo-tryptophan, p-hydroxyphenylacetic
acid (pHA), and pHA adducts of lysine or lipids.

[0031]A sixth aspect of the invention provides a kit for measuring the
levels of oxidized high density lipoprotein (HDL) products. Such a kit
may comprise one or more of a buffer, an antibody, a chemical reagent and
a positive control for one or more oxidized high density lipoprotein
(HDL) products.

[0033]FIG. 2 describes the effect of C- or taurine on HDL nitration
by the myeloperoxidase-H2O2--NO2- system or
HOCl--NO2-. HDL was exposed for 60 min at 37° C. to
myeloperoxidase in phosphate buffer supplemented with 250 μM
H2O2, 500 μM NO2- and (A) the indicated
concentration of Cl- or (B) the indicated concentration of taurine
and 100 mM Cl-. (C) HDL was exposed for 60 min at 37° C. in
phosphate buffer containing 500 μM NO2- and the indicated
concentration of HOCl.

[0036]FIG. 5 demonstrates the mass spectrometric quantification of
3-nitrotyrosine in HDL isolated from plasma and human atherosclerotic
lesions. Plasma was obtained from healthy humans and humans with
established coronary artery disease. Human atherosclerotic tissue was
obtained at surgery from subjects undergoing carotid endarterectomy. HDL
was isolated from plasma and atherosclerotic aorta by sequential
ultracentrifugation. 13C-Labeled internal standards were added, and
the protein was hydrolyzed with acid. Derivatives of the oxidized amino
acids were quantified by isotope dilution negative-ion electron capture
GC/MS with selected ion monitoring.

[0037]FIG. 6 demonstrates the association between 3-nitrotyrosine and
3-chlorotyrosine levels in HDL isolated from human atherosclerotic
lesions or plasma. Levels of oxidized amino acids in HDL were determined
in lesion HDL and circulating HDL as described in the legend to FIG. 5.

[0043]FIG. 12 depicts dityrosine levels present in the urine in control
patients and diabetic patients having undergone a renal transplant. The
urinary dityrosine levels in nmol/mol creatinine are elevated about 50%
in the diabetic patients having undergone a renal transplant.

[0044]FIG. 13 depicts nitrotyrosine levels present in the plasma in
control patients and diabetic patients having undergone a renal
transplant. The plasma nitrotyrosine levels in nmol/mol tyrosine are
elevated about 100% in the diabetic patients having undergone a renal
transplant as compared to control patients. HDL was isolated from the
plasma by sequential ultracentrifiguation. 13C-labeled internal
standards were added, and the protein was hydrolyzed with.acid.
Derivatives of the oxidized amino acids were quantified by isotope
dilution negative-ion electron capture GC/MS with selected monitoring.
Results are normalized to the protein content of L-tyrosine, the
precursor of 3-nitrotyrosine and 3-chlorotyrosine.

[0045]FIG. 14 depicts the correlation between nitrotyrosine levels present
in the plasma in control patients and diabetic patients having undergone
a renal transplant and levels of Hemoglobin A1C. The plasma nitrotyrosine
levels are presented in umol/mol tyrosine.

[0046]FIG. 15 depicts myeloperoxidase levels present in the plasma in
control patients and diabetic patients having undergone a renal
transplant. The plasma myeloperoxidase levels in pM are elevated in the
diabetic patients having undergone a renal transplant as compared to
control patients.

[0047]FIG. 16 depicts the correlation between dityrosine levels present in
the urine in control patients and diabetic patients having undergone a
renal transplant and levels of Hemoglobin A1C.

[0048]FIG. 17 depicts the correlation between nitrotyrosine levels present
in the plasma in control patients and diabetic patients having undergone
a renal transplant and levels of myeloperoxidase present in the plasma of
the same patients.

[0049]FIG. 18 depicts the correlation between nitrotyrosine levels present
in the plasma in control patients and diabetic patients having undergone
a renal transplant and levels of myeloperoxidase present in the plasma of
the same patients.

[0050]FIG. 19 depicts the correlation between dityrosine levels present in
the urine in control patients and diabetic patients having undergone a
renal transplant and levels of nitrotyrosine present in the plasma of the
same patients.

[0051]FIG. 20 demonstrates that the levels of nitrotyrosine and
chlorotyrosine, respectively represented in μmol of each per mol of
tyrosine, are elevated in HDL isolated from atherosclerotic tissue in
diabetic patients as compared to control patients.

DETAILED DESCRIPTION

[0052]Before the present methods and treatment methodology are described,
it is to be understood that this invention is not limited to particular
methods, and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the terminology
used herein is for purposes of describing particular embodiments only,
and is not intended to be limiting, since the scope of the present
invention will be limited only in the appended claims.

[0053]As used in this specification and the appended claims, the singular
forms "a", "an", and "the" include plural references unless the context
clearly dictates otherwise. Thus, for example, references to "the method"
includes one or more methods, and/or steps of the type described herein
and/or which will become apparent to those persons skilled in the art
upon reading this disclosure and so forth.

[0054]Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although any methods
and materials similar or equivalent to those described herein can be used
in the practice or testing of the invention, the particular methods and
materials are now described. All publications mentioned herein are
incorporated herein by reference.

Definitions

[0055]The terms used herein have the meanings recognized and known to
those of skill in the art; however, for convenience and completeness,
particular terms and their meanings are set forth below.

[0056]"Treatment" refers to the administration of a drug or the
performance of procedures with respect to a subject, for either
prophylaxis (prevention) or to cure the infirmity or malady in the
instance where the subject is afflicted.

[0057]As used herein, "assessing" refers to determining whether an
individual is at risk or susceptible to developing a disease or
pathological condition caused in part by abnormal levels of one or more
oxidized high density lipoprotein (HDL) product. The condition may be any
in which there exists a higher than normal level of oxidative stress
compounds, such as those described in the present invention. However, one
particular condition for which a correlation has been made is
cardiovascular disease. A determination may be made based on the
particular disease and symptoms associated with the disease, and whether
or not the cause of the disease or condition may be attributed, at least
in part, to high levels of oxidation of cells, tissues, proteins or other
molecular or chemical entities which are candidates for damage caused by
oxidative stress, as evidenced by high levels of one or more oxidized
high density lipoprotein (HDL) products.

[0058]By "individual" or "patient" or "subject" is meant a human or
non-human mammal that may benefit from the diagnostic tests or methods
described in the present application, for example, an individual at risk
for developing or having a cardiovascular disease or one at risk for
having a heart attack. Alternatively, other individuals may be
predisposed to diseases or conditions other than cardiovascular disease,
caused by high levels of oxidative stress. Accordingly, the individual
may be treated prophylactically with agents appropriate for the specific
disease. For example, in the case of cardiovascular disease, the
individual may be required to alter their life style such that a strict
regimen of diet and exercise may be necessary to stabilize their
condition.

[0059]"Surrogate biomarker" or "biomarker" or "marker" as used herein,
refers to a highly specific molecule, the existence and levels of which
are causally connected to a complex biological process, and reliably
captures the state of the process. Furthermore, a surrogate biomarker, or
marker, to be of practical importance, must be present in samples that
can be obtained from individuals without endangering their physical
integrity or well-being, preferentially from biological fluids such as
blood, plasma, urine, saliva, CSF or tears. While the markers of
oxidative damage include the products of oxidative stress, such as
increased lipid peroxides, decreased glutathione, or dityrosine,
nitrotyrosine, dinitrotyrosine, 3-chlorotyrosine, nitrophenyl alanine,
chlorophenyl alanine, and the levels of these biomarkers should reflect
the degree of oxidative stress in the body as a result of certain
diseases or conditions, it is to be understood that measuring the levels
of enzymes responsible for generation of these products is also useful
for assessing the risk factors for development of certain diseases, as
described herein. Thus, the oxidized high density lipoprotein (HDL)
products can also be considered as markers of the disease process, or
risk prognosticators, especially in cardiovascular disease, since there
is an elevation in oxidized high density lipoprotein (HDL) products in
patients suffering from CVD, or at risk for developing CVD. Furthermore,
the presence of these biomarkers should reflect the need for either
prophylactic therapy, or for a need for possible future therapy with
appropriate cardiovascular drugs. Alternatively, when the levels of these
two markers fall outside of the normal range, a patient may be put on a
regimen of diet and exercise until the level of markers normalizes. The
normalization of these markers as well as normalization of the levels of
other tests commonly used to diagnose CVD should also reflect the
efficiency of therapy if a patient is undergoing such therapy.

[0060]By "efficacy" is meant whether the treatment results in a desired
outcome. For example, in the case of treating a patient having high
levels of oxidized high density lipoprotein (HDL) products, an increase
in the amount of atherosclerotic plaque which ultimately may lead to
progressive cardiovascular disease correlates with an increased level of
the subject HDL oxidation products. A desired outcome is therefore
reduction in the levels of HDL oxidation products.

[0061]The "reference range", as used herein, can be determined by one
skilled in the art using the methods described herein by a laboratory
that can establish a range of levels of oxidized high density lipoprotein
(HDL) that are characteristic for either an individual free of, or not
susceptible to, a pathological condition, such as a cardiovascular
disease, or who are not predisposed for having progressive cardiovascular
disease or further sequelae therefrom, and establishing the range of
oxidized high density lipoprotein (HDL) in a subject prone to such
conditions. This "reference range" may be used in the methods of the
present invention for comparative purposes when testing a patient for the
presence of or the susceptibility to acquiring such conditions as
outlined herein. Based on this comparison, a conclusion may be drawn as
to whether a pathological condition, such as a cardiovascular disease, is
present in the subject being tested. Those skilled in the art will
appreciate how to establish a cut-off value suitable for differentiating
subjects suffering from such conditions from subjects not suffering from
such conditions.

[0062]"Vulnerable plaque" is a type of fatty buildup in an artery thought
to be caused by inflammation. The plaque is covered by a thin, fibrous
cap that upon rupture may lead to the formation of a blood clot and,
ultimately, occlusion of the artery. Plaque rupture most often occurs in
smaller arteries, such as the coronary arteries, which supply blood to
the heart muscle. The occlusion of a coronary artery can lead to a heart
attack. Even moderately occluded arteries with areas of vulnerable plaque
are also likely to lead to a heart attack.

General Description

[0063]The present invention relates to diagnostic tests and methods to
better identify those subjects having, or at risk for developing, a
pathological condition associated with abnormal levels of one or more
oxidized high density lipoprotein (HDL) products, in particular,
cardiovascular disease.

[0064]Oxidative stress has been implicated in a number of pathological
disease processes, including atherosclerosis (Makela R. et al. (2003) Lab
Invest 83(7):919-25). Oxidative stress may be defined as an imbalance
between the production and degradation of reactive oxygen species such as
superoxide anion, hydrogen peroxide, lipid peroxides, and peroxynitrite.
Enzymatic degradation of these reactive oxygen species is achieved
primarily by the enzymes glutathione peroxidase, superoxide dismutase and
catalase (Forsberg et al. (2001) Arch Biochem Biophys 389: 84-93).

[0065]The glutathione/glutathione peroxidase system is one of the primary
antioxidant defense systems in mammals. Glutathione peroxidase 1 is the
key antioxidant enzyme in most cells, and this enzyme uses glutathione to
reduce hydrogen peroxide to water and lipid peroxides to their respective
alcohols ((Flohe, L. (1988), Basic Life Sci 57: 1825-35). Mice having a
deficiency in this enzyme demonstrate abnormal vascular and cardiac
function and structure (Forgione, M. et al. (2002), 106:1154-8). More
recent studies in humans by Blankenberg et al. have shown that a low
level of activity of this enzyme (GPX) is associated with an increased
risk of cardiovascular events (Blankenberg, S, et al. (2003), N. England
J. Med. 349: 1605-1613).

[0066]Currently, several of the known risk factors for cardiovascular
disease are used by physicians in risk prediction algorithms in an
attempt to target those individuals who are at highest risk for
development of CVD. If an individual presents with a high-risk profile,
the individual may be placed on appropriate therapy to address those
factors that can be controlled or modified. Other risk factors associated
with CVD may be addressed by simple changes in lifestyle thereby allowing
these individuals to modify certain factors to lower their risk profile,
e.g., changes in diet or exercise. There is a need for expanding such
algorithms to take into account other factors that should be included in
a patient's risk profile for development of CVD.

[0067]Accordingly, the present invention provides a multidimensional and
comprehensive method for assessing an individual's risk for developing
diseases associated with high levels of oxidative stress-induced
compounds, such as CVD. Previous tests for measuring an individual's
level of oxidative stress have relied primarily on the measurement of one
primary marker of oxidative stress, such as lipid peroxides. The present
invention provides for the quantitation of oxidized high density
lipoprotein (HDL) products. The present invention will thus provide for
the interrelationship between disease risk or state and oxidized high
density lipoprotein (HDL) products.

[0068]It is a further object of the present invention to be able to
measure the efficacy of therapy once an individual has started therapy
with agents known to those skilled in the art. The results, when combined
with other risk factors for the specific disease, such as, but not
limited to CVD, aid in assessing an individual's potential susceptibility
for these diseases, which result in part from an increase in oxidized
high density lipoprotein (HDL) products.

[0069]The "reference range" for oxidized high density lipoprotein (HDL)
products, as used herein, can be determined by one skilled in the art
using the methods described herein. A laboratory can establish a range of
levels of oxidized high density lipoprotein (HDL) products that are
characteristic for either an individual free of, or not susceptible to, a
pathological condition, such as a cardiovascular disease, or who are not
predisposed for progressive disease or sequelae such as heart attack, and
can also establish a range of oxidized high density lipoprotein (HDL)
products in a subject prone to such conditions by measuring one or more
oxidized high density lipoprotein (HDL) products in these patient
populations. Furthermore, these values may be used in conjunction with
other standard tests used to assess a patient's risk profile for
developing cardiovascular disease, such as, but not limited to, standard
blood chemistry tests for measuring levels of LDL, HDL, triglycerides,
cholesterol and the like. The "reference range" may then be used in the
methods of the present invention for comparative purposes when testing a
patient for the presence of or the susceptibility to acquiring such
conditions as outlined herein. Based on this comparison, a conclusion can
be drawn as to whether a pathological condition, such as a cardiovascular
disease, is present in the individual being tested. Those skilled in the
art may routinely establish cut-off values suitable for differentiating
individuals suffering from such conditions from individuals not suffering
from such conditions.

Providing a Biological Sample for Use in the Methods of the Present
Invention

[0070]In particular embodiments the assays are performed using a
biological sample from the individual of interest. While the assays are
applicable in humans, they are not so limited. It is believed similar
oxidative damage exists essentially in all mammals and thus the assays of
this invention are contemplated for veterinary applications as well.
Thus, suitable individuals include, but are not limited to humans,
non-human primates, canines, equines, felines, porcines, ungulates,
lagomorphs, and the like.

[0071]A suitable biological sample includes a sample of a biological
material, which may be selected from a whole blood sample or a derivative
thereof. As used herein a blood sample includes a sample of whole blood,
blood cells or a blood fraction (e.g. serum or plasma). The cells may be
separated out into erythrocytes, white blood cells including monocytes,
PMNs, lymphocytes and may be used as whole cells or cell lysates may be
prepared. The sample may be fresh blood or stored blood (e.g. in a blood
bank) or blood fractions. The sample may be a blood sample expressly
obtained for the assays of this invention or a blood sample obtained for
another purpose, which can be subsampled for the assays of this
invention. In another embodiment, the bodily sample may be saliva or CSF.

[0072]The sample may be pre-treated as necessary by dilution in an
appropriate buffer solution, heparinized, concentrated if desired, or
fractionated by any number of methods including but not limited to
ultracentrifugation, fractionation by fast performance liquid
chromatography (FPLC), or precipitation of proteins with dextran sulfate
or other methods. Any of a number of standard aqueous buffer solutions,
employing one of a variety of buffers, such as phosphate, Tris, or the
like, at physiological pH can be used.

Assay Formats

[0073]The methods of this invention may use assays, which may be
practiced, in almost a limitless variety of formats depending on the
particular needs at hand. Such formats include, but are not limited to
traditional "wet chemistry" (e.g. as might be performed in a research
laboratory), high-throughput assay formats (e.g. as might be performed in
a pathology or other clinical laboratory), and "test strip" formats,
(e.g. as might be performed at home or in a doctor's office).

Traditional Wet Chemistry

[0074]The assays of this invention can be performed using traditional "wet
chemistry" approaches. Basically this involves performing the assays as
they would be performed in a research laboratory. Typically the assays
are run in a fluid phase (e.g. in a buffer with appropriate reagents
(e.g. lipids, oxidized lipids, oxidizing agent, etc.) added to the
reaction mixture as necessary. The oxidized lipid concentrations are
assayed using standard procedures and instruments, e.g. as described in
the examples.

High-Throughput Assay Formats

[0075]Where population studies are being performed, and/or in
clinical/commercial laboratories where tens, hundreds or even thousands
of samples are being processed (sometimes in a single day) it is often
preferably to perform the assays using high-throughput formats. High
throughput assay modalities are highly instrumented assays that minimize
human intervention in sample processing, running of the assay, acquiring
assay data, and (often) analyzing results. In particular embodiments,
high throughput systems are designed as continuous "flow-through"
systems, and/or as highly parallel systems.

[0076]Flow through systems typically provide a continuous fluid path with
various reagents/operations localized at different locations along the
path. Thus, for example a blood sample may be applied to a sample
receiving area where it is mixed with a buffer, the path may then lead to
a cell sorter that removes large particulate matter (e.g. cells), the
resulting fluid may then flow past various reagents (e.g. where the
reagents are added at "input stations" or are simply affixed to the wall
of the channel through which the fluid flows. Thus, for example, the
sample may be sequentially combined with a lipid (e.g. provided as an
LDL), then an oxidation agent, an agent for detecting oxidation, and a
detector where a signal (e.g. a calorimetric or fluorescent signal) is
read providing a measurement of oxidized lipid.

[0077]In highly parallel high throughput systems samples are typically
processed in microtiter plate formats (e.g. 96 well plates, 1536 well
plates, etc.) with computer-controlled robotics regulating sample
processing reagent handling and data acquisition. In such assays, the
various reagents may all be provided in solution. Alternatively some or
all of the reagents (e.g. oxidized lipids, indicators, oxidizing agents,
etc.) may be provided affixed to the walls of the microtiter plates.

[0078]High throughput screening systems that can be readily adapted to the
assays of this invention are commercially available (see, e.g., Zymark
Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman
Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,
Mass., etc.). These systems typically automate entire procedures
including all sample and reagent pipetting, liquid dispensing, timed
incubations, and final readings of the microplate in detector(s)
appropriate for the assay. These configurable systems provide high
throughput and rapid start up as well as a high degree of flexibility and
customization. The manufacturers of such systems provide detailed
protocols. Thus, for example, Zymark Corp. provides technical bulletins
describing screening systems for detecting the modulation of gene
transcription, ligand binding, and the like.

"Test Strip" Assay Formats

[0079]The methods of the present invention may also utilize assays which
are provided in "test well" or "test strip" formats. In "test well" or
"test strip" formats, the biological sample is typically placed in the
well or applied to a receiving zone on the strip and then a fluorescent
or calorimetric indicator appears which, in this case, provides a measure
of the level of the enzymes present or absent from the sample.

[0080]Many patents have been issued which describe the various physical
arrangements for blood testing. These include systems that involve
lateral or horizontal movement of the blood, as well as plasma testing.
For example, U.S. Pat. Nos. 4,876,067, 4,861,712, 4,839,297, and
4,786,603 describe test carriers and methods for analytical determination
of components of bodily fluids, including separating plasma from blood
using glass fibers and the like. These patents, all teach systems which
require some type of rotation of test pads or a portion of the test pads
during use. U.S. Pat. No. 4,816,224 describes a device for separating
plasma or serum from whole blood and analyzing the serum using a glass
fiber layer having specific dimensions and absorption to separate out the
plasma from the whole blood for subsequent reaction. Similarly, U.S. Pat.
No. 4,857,453 describes a device for performing an assay using capillary
action and a test strip containing sealed liquid reagents including
visible indicators. U.S. Pat. No. 4,906,439 describes a diagnostic device
for efficiently and accurately analyzing a sample of bodily fluid using
fluid delivery in a lateral movement via flow through channels or
grooves.

[0081]Oxidized high density lipoprotein (HDL) products may be measured on
the basis of their biological or chemical activity and/or their mass. The
following describes methods for such measurements.

[0082]Buss et al. noted that 3-chlorotyrosine, a specific biomarker of the
neutrophil oxidant, hypochlorous acid, was present in higher quantities
in tracheal aspirates of preterm infants compared to infants having
normal birth weights without respiratory distress. The level of this
marker correlated strongly with myeloperoxidase activity. These studies
support a role for neutrophil oxidants in the pathology of chronic lung
disease. Shishehbor et al. have also done studies that demonstrate that
nitrotyrosine, a specific marker for protein modification by nitric oxide
derived oxidants, is enriched in atherosclerotic lesions and in low
density lipoprotein derived from human atheromas (Shishehbor et al.
(2003), JAMA, 289(13): 1675-80). Yet further evidence for the role of the
MPO/H2O2/halide system in human atheroslerotic lesions has been
demonstrated by Malle et al. (2000), Eur. J. Biochem. 267: 4495-4503).
Specific quantitative methods for detecting 3-chlorotyrosine,
3-bromotyrosine and 3-nitrotyrosine have been elucidated by Gaut et al.
(Anal. Biochem. (2002), 300: 252-259). Specific quantitative methods for
detecting 3-chlorotyrosine, 3-bromotyrosine and 3-nitrotyrosine have been
elucidated by Gaut et al. (Anal. Biochem. (2002), 300: 252-259). However,
PCT publication number WO9604311 discloses a monoclonal antibody to
nitrotyrosine, thus providing the means for development of immunological
assays for measuring this marker for oxidative damage. Another antibody
to nitrotyrosine can be found in the Oxis International catalog, number
24312. Furthermore, an assay to measure nitrotyrosine is provided for by
Oxis International in the BIOXYTECH®Nitrotyrosine-EIA kit (Catalog
Number 21055).

Kits

[0083]The diagnostic tests and methods of the present invention provide
for measuring the amounts of oxidized high density lipoprotein (HDL)
products as a means of assessing the risk of an individual for having or
developing a condition associated with high levels of oxidative
stress-induced products, such as CVD. In a particular embodiment, one or
more oxidized high density lipoprotein (HDL) products are quantitated
using standard reagents and kits, which are commercially available to
measure each marker individually (See above). Thus, the present invention
provides a quantitative and accurate means of assessing a subject's need
for antioxidative therapy, or therapy with agents that are standardly
used to treat CVD, by measuring all of these parameters. To the
inventor's knowledge, no other art currently exists which describes
combining the concurrent non-invasive techniques and measurements
described herein for assessing a subject's risk for developing CVD.

[0084]While the kits described above provide the accuracy and sensitivity
necessary for measurements of oxidized high density lipoprotein (HDL)
products as described in the present invention, further kits may be
developed that contain the antibodies, reagents, buffers, standards and
instructions for assaying both enzymes using the same format, e.g. ELISA,
or a colorimetric assay. The test kits would be modified appropriately
depending on whether the samples to be assayed consist of whole cells,
cell lysates or a combination thereof.

[0085]In some embodiments, an assay format is provided in which binding
partners such as antibodies can be obtained or prepared for the oxidized
high density lipoprotein (HDL) products. Biotin-avidin,
biotin-streptavidin or other biotin-binding-reagent reactions can be used
to enhance or modulate the test. However, any such assay can be devised
using other binding partners to the analytes, including but not limited
to extracellular or intracellular receptor proteins which recognize the
analytes, binding fragments thereof, hybridization probes for nucleic
acids, lectins for carbohydrates, etc. The particular selection of
binding partners is not limiting, provided that the binding partners
permit the test to operate as described herein. The preselected analytes,
when present, are detectable by binding two binding partners, one
immobilized on the test strip (or whatever format the assay is provided)
and another part of a conjugate. This is taken into consideration in the
selection of the reagents for the assay.

[0086]If a dry test strip is desired, this may be set up in any format in
which contact of the sample with the reagents is permitted and the
formation and mobility of the immunocomplexes and other complexes forming
therein are permitted to flow and contact an immobilized reagent at the
capture line. Various formats are available to achieve this purpose,
which may be selected by the skilled artisan.

[0087]The label portion of the mobile, labeled antibody to the marker may
be a visible label, such as gold or latex, an ultraviolet absorptive
marker, fluorescent marker, radionuclide or radioisotope-containing
marker, an enzymatic marker, or any other detectable label. A visibly
detectable marker or one that can be easily read in a reflectometer is
preferred, for use by eye, reading or confirmation with a reflectometer.
Other labels may be applicable to other semi-automated or automated
instrumentation.

[0088]The conjugates of the invention may be prepared by conventional
methods, such as by activation of an active moiety, use of
homobifunctional or heterobifunctional cross-linking reagents,
carbodiimides, and others known in the art. Preparation of, for example,
a gold-labeled antibody, a conjugate between an antibody and an analyte
(not an immunocomplex but a covalent attachment which allows each member
to independently exhibit its binding properties), biotinylation of an
antibody, conjugation of streptavidin with a protein, immobilization of
antibodies on membrane surfaces, etc., are all methods known to one of
skill in the art.

[0089]A kit may have at least one reagent for carrying out an assay of the
invention, such as a kit comprising a conjugate between a biotin-binding
reagent and an antibody to an oxidized high density lipoprotein (HDL)
product. Preferably, the kit comprises all of the reagents needed to
carry out any one of the aforementioned assays, whether it be
homogeneous, heterogeneous, comprise a single conjugate of the marker
conjugated to an antibody to the analyte, or comprise two reagents which
serve this function (such as a biotinylated antibody to the analyte plus
a streptavidin-marker conjugate, or a biotinylated marker plus a
streptavidin conjugated to an antibody to the analyte conjugate), or
whether the assay employs an immobilized antibody to the analyte and a
labeled antibody to a different site on the analyte. Referring to the
first analyte as analyte and the second analyte as marker, and a second
binding partner as a binding partner which recognizes a different epitope
than the first binding partner mentioned, the kits are non-limiting
examples of those embraced herein.

[0090]In the foregoing kits, the binding partners are preferably
antibodies or binding portions thereof, and both the binding partner to
the analyte (the oxidized high density lipoprotein (HDL) products) and
the second binding partner to the analytes capable of simultaneously
binding to the analyte. The immobilized binding partner may be provided
in the form of a capture line on a test strip, or it may be in the form
of a microplate well surface or plastic bead. The kits may be used in a
homogeneous format, wherein all reagents are added to the sample
simultaneously and no washing step is required for a readout, or the kits
may be used in a multi-step procedure where successive additions or steps
are carried out, with the immobilized reagent added last, with an
optional washing step.

[0091]The antibodies specific for the two markers may be obtained
commercially, or can be produced by techniques known to those skilled in
the art.

Nitro Oxidized HDL Products

[0092]NO produced by endothelial cells regulates vasomotor tone and
inhibits smooth muscle cell proliferation and leukocyte adhesion (Moncada
et al., (1991) Pharmacological Reviews 43, 109-142). The larger amounts
produced by macrophages help kill microbes and tumor cells. Under
pathological conditions, however, reactive nitrogen species derived from
NO may injure vascular tissue (Beckman et al., (1996) Am J Physiol 271,
C1424-1437). One important pathway may be the rapid reaction of NO with
superoxide, which may simultaneously create a deficit in the amount of NO
needed for normal physiology and generate the potent oxidizing
intermediate ONOO-. Id. Overproduction of superoxide by phagocyte
and nonphagocyte NADPH oxidases (such as the NOX family of enzymes) and
dysregulation of NO synthase might contribute to this pathway (Babior et
al., (2002) Arch Biochem Biophys 397, 342-344; Chen et al., (2003) Free
Radic Biol Med 35, 117-132). Moreover, myeloperoxidase, which is enriched
in human atherosclerotic lesions (Daugherty et al., (1994) Journal of
Clinical Investigation 94, 437-444; Sugiyama et al., (2001) Am J Pathol
158, 879-891), uses NO2- derived from NO to generate reactive
intermediates that form 3-nitrotyrosine in proteins in vitro (Eiserich et
al., (1998) Nature 391, 393-397; van der Vliet et al., (1997) J Biol Chem
272, 7617-7625). They also peroxidize the lipid moieties of LDL,
converting the lipoprotein to a form that is recognized by the macrophage
scavenger receptor (Podrez et al., (1999) J Clin Invest 103, 1547-1560).
Unregulated uptake of such modified lipoprotein may play a role in
cholesterol accumulation by macrophages, a critical early step in
atherogenesis.

[0093]The present invention demonstrates that HDL is oxidized by reactive
nitrogen species in vivo. The data demonstrate a 5-fold higher level of
3-nitrotyrosine, a specific marker for reactive nitrogen intermediates,
in HDL isolated from atherosclerotic tissue than in circulating HDL. The
level of 3-nitrotyrosine in lesion HDL is similar to those previously
reported for lesion LDL (Leeuwenburgh et al., (1997) Journal of
Biological Chemistry 272, 1433-1436), indicating that both lipoproteins
are nitrated to a similar extent in the human artery wall.

[0094]In immunohistochemical studies of atherosclerotic lesions,
myeloperoxidase is found to co-localize with epitopes recognized by
antibodies to 3-nitrotyrosine, suggesting that it is an important source
of reactive nitrogen species in the artery wall. However, there is no
significant correlation between levels of 3-nitrotyrosine and
3-chlorotyrosine, a specific product of myeloperoxidase (Gaut et al.,
(2001) Proc Natl Acad Sci USA 98, 11961-11966), in HDL isolated from
atherosclerotic lesions, suggesting that pathways independent of
myeloperoxidase also nitrate HDL in the artery wall. Alternatively,
macrophage scavenger receptors might bind and internalize chlorinated HDL
and nitrated HDL at different rates, altering their relative
concentrations in lesion HDL (Heinecke, (2002) Free Radic Biol Med 32,
1090-1101). It is also possible that chlorinated HDL and nitrated HDL are
extracted with different efficiencies from vascular tissue. Nitrated HDL
may represent a previously unsuspected biochemical link between
inflammation, nitrosative stress, and atherogenesis.

[0095]The data provided herein also demonstrate that circulating HDL is
nitrated on tyrosine residues. Importantly, HDL's content of
3-nitrotyrosine is twice as high in humans with established coronary
artery disease as in healthy subjects.

[0096]Myeloperoxidase is likely to use NO2- as a physiological
substrate when it generates reactive nitrogen species.
Myeloperoxidase-deficient mice have a markedly lower level of free
3-nitrotyrosine than wild-type mice after intraperitoneal infection with
bacteria (Gaut et al., (2002) J Clin Invest 109, 1311-1319). In contrast,
the two strains have comparable levels of the nitrated amino acid when
peritoneal inflammation is induced by cecal ligation and puncture.
Although both models of intraabdominal inflammation produce an intense
neutrophil response and a marked increase in the level of
3-chlorotyrosine, they differ in one important respect: levels of
NO2- and NO3- were 20-fold higher in mice infected
intraperitoneally with bacteria than in mice subjected to cecal ligation
and puncture (Gaut et al., (2002) J Clin Invest 109, 1311-1319). These
results indicate that myeloperoxidase in vivo generates oxidants that can
nitrate tyrosine. They also suggest that the enzyme produces these
oxidants only when levels of NO2- and NO3- increase
substantially.

[0097]Collectively, the data provided herein indicate that reactive
nitrogen species oxidize HDL in the human artery wall. Nitrated HDL also
circulates in blood, and individuals suffering from clinically
significant atherosclerosis contain elevated levels of the oxidized
lipoprotein in their plasma.

Chloro Oxidized High Density Lipoprotein (HDL) Products

[0098]The level of 3-chlorotyrosine in HDL isolated from human
atherosclerotic lesions was 6-fold higher than that in circulating HDL
from human subjects. Moreover, the level of 3-chlorotyrosine was 8-fold
higher in HDL isolated from plasma of subjects with coronary artery
disease than in HDL from plasma of healthy subjects. Hence, HOCl derived
from myeloperoxidase contributes to HDL oxidation in the artery wall.
Elevated levels of 3-chlorotyrosine in circulating HDL represents a novel
marker for clinically significant atherosclerosis.

[0099]HDL and lipid-free apo A-I oxidized by HOCl are less able to remove
cholesterol from cells by the ABCA1 pathway than native HDL and apo A-I.
Because HDL contains both phospholipids and apolipoproteins, it can
remove cellular cholesterol by both ABCA1-independent and -dependent
mechanisms. Treating HDL with HOCl does not inhibit cholesterol efflux by
ABCA1-independent processes but significantly reduces efflux from
ABCA1-expressing cells. Similarly, oxidizing lipid-free apo A-I (which
removes cellular lipids exclusively by the ABCA1 pathway) with HOCl
markedly reduces cholesterol efflux. This inhibitory effect is near
maximal when HOCl has chlorinated ˜50% of the tyrosine residues in
apo A-I. In contrast, treating HDL or apo A-I with hydrogen peroxide,
which selectively oxidizes methionines, does not affect cholesterol
efflux. Previous studies have shown that methionine oxidation fails to
alter apo A-I-promoted cholesterol efflux from cultured cells (Panzenbock
et al., 2000. J Biol Chem 275:19536-19544). HOCl oxidation of an
apolipoprotein-mimetic amphipathic α-helical peptide reduced its
ability to remove cellular cholesterol. Thus, myeloperoxidase-mediated
chlorination of tyrosine residues in HDL apolipoproteins in the artery
wall may impair cholesterol removal and enhance atherogenesis.

[0100]The primary e amino group of lysine facilitates the regioselective
chlorination of tyrosine residues in the YxxK motif of apo A-I and
synthetic peptides by a pathway involving a chloramine intermediate.
Modeling and structural studies indicate that tyrosine and lysine
residues separated by two amino acids are adjacent on the same face of an
α-helix, suggesting that the YxxK motif could direct protein
chlorination if it resided in an α-helix. A single tyrosine residue
in the 8th amphipathic α-helix of apolipoprotein A-I was the
major site of chlorination by HOCl and that this tyrosine resides in the
YxxK motif (Bergt et al., 2004. J Biol Chem 279:7856-7866).

[0101]The data described herein demonstrate that oxidative species
generated by phagocytes chlorinate specific tyrosine residues in apo A-I.
Modification of these residues impairs the protein's ability to promote
cholesterol efflux from lipid-laden macrophages, contributing to the
formation of atherosclerotic lesions. Because phagocytes store NADPH
oxidase and myeloperoxidase in their plasma membrane and secretory
compartments, respectively, oxidation is likely to be tightly restricted
in space by local changes in oxidant concentrations. It is important to
note that apo A-I promotes cholesterol efflux from cells by interacting
with ABCA1 at the plasma membrane of macrophages. Local, pericellular
production of oxidants by phagocytes is a physiological mechanism for
oxidizing apo A-I and inhibiting HDL function during atherogenesis.
Moreover, 3-chlorotyrosine in HDL protein may serve as a molecular
fingerprint for the pathway that mediates oxidative damage in patients
suffering from coronary artery disease.

[0103]Isolation of HDL. Blood anticoagulated with EDTA was collected from
healthy adults and patients with clinically and angiographically
documented coronary artery disease who had fasted overnight. HDL
(d=1.125-1.210 g/mL) was prepared from plasma by sequential
ultracentrifugation. Isolated HDL was depleted of apo E and apo B100 by
heparin-agarose chromatography (Mendez et al., (1991) J Biol Chem 266,
10104-10111). The Human Studies Committees at University of Washington
School of Medicine and Wake Forest University School of Medicine approved
all protocols involving human material.

[0104]Isolation of lesion HDL. Atherosclerotic tissue was harvested at
endarterectomy, snap frozen, and stored frozen at -80° C. until
analysis. Lesions from a single individual (˜0.5 g wet weight) were
mixed with dry ice and pulverized in a stainless steel mortar and pestle.
All subsequent procedures were carried out at 4° C. Powdered
tissue was suspended in 2 mL of antioxidant buffer A (138 mM NaCl, 2.7 mM
KCl, 100 μM diethylenetriaminepentaacetic acid (DTPA), 100 μM
butylated hydroxyl toluene (BHT), protease inhibitor cocktail (Roche
Diagnostics, Mannheim, Germany), 10 mM sodium phosphate, pH 7.4) in a 2
mL centrifuge tube and rocked gently overnight. Tissue was removed by
centrifugation, the supernatant was collected, and the pellet was
extracted a second time with antioxidant buffer for 1 h. The pooled
supernatants were centrifuged at 100,000×g for 30 min, and the
pellet and uppermost lipemic layer were discarded.

[0105]HDL was isolated from the tissue extract by sequential density
ultracentrifugation (d=1.063-1.210 g/mL; (47)). DTPA and BHT (each 100
μM) were included in all solutions used for lipoprotein isolation.
Lesion HDL was equilibrated with buffer A (0.1 mM DTPA, 100 mM sodium
phosphate, pH 7.4) using a 100 kDa cut-off filter device (Millipore,
Bredford, Mass.). Apo A-I in lesion HDL was detected by Western blotting
using a rabbit IgG polyclonal antibody to human apo A-I (Calbiochem, La
Jolla, Calif.) followed by a horseradish peroxidase-conjugated goat
anti-rabbit IgG and enhanced chemiluminescence detection. Protein was
determined using the Lowry assay, with albumin as the standard (BioRad;
Hercules, Calif.).

[0109]Isotope dilution GC/MS Analysis. All samples were manually injected
using an on column injector and a Hewlett Packard 6890 gas chromatograph
equipped with a 15 m DB-5 capillary column (0.25 mm id, 0.33 micron film
thickness, J & W Scientific) interfaced with a Hewlett Packard 5973 mass
detector. The t-butyl dimethylsilyl derivatives of amino acids were
quantified by selected ion monitoring, using isotope dilution
negative-ion chemical ionization GC/MS (Gaut et al., (2001) Proc Natl
Acad Sci USA 98, 11961-11966; Gaut et al., (2002) Anal Biochem 300,
252-259). The level of 3-nitrotyrosine was quantified using the ratio
between the ion of m/z 518 derived from 3-nitrotyrosine
([M-O-t-butyl-dimethylsilyl]-) and the ion of m/z 524 derived from
3-nitro[13C6]tyrosine. The level of 3-chlorotyrosine was
quantified using the ratio between the ion of m/z 489 derived from
3-chlorotyrosine ([M-Cl-t-butyl-dimethylsilyl]-) and the ion of m/z
495 derived from 3-chloro[13C6]chlorotyrosine. Potential
artifact formation was monitored as the appearance of ions at m/z 528
(nitration) or m/z 499 (chlorination) derived from L-[13C9,
15N]tyrosine added prior to sample work-up. Under these experimental
conditions, artifact formation was <20% of 3-nitrotyrosine and <5%
of 3-chlorotyrosine. L-Tyrosine is present at 10,000-fold higher levels
than the oxidation products. Therefore the sample was diluted 1:100 and
analyzed in a separate injection. L-Tyrosine and
L-[13C6]tyrosine were quantified using the ions
([M-CO2-t-butyl-dimethylsilyl]-) at m/z 407 and m/z 413,
respectively. Under these chromatography conditions, authentic products
and isotopically labeled standards were baseline-separated and exhibited
retention times identical to those of analytes derived from tissue
samples. The limit of detection (signal/noise>10) was <1 femtomol
for all the amino acids.

[0110]Statistical analysis. Results represent means±SEM. Differences
between two groups were compared using an unpaired Student's t-test.
Correlations were determined using linear regression analysis for
nonparametric data (Sigma Stat, SPSS). A P value<0.05 was considered
significant.

Results

[0111]Myeloperoxidase generates 3-nitrotyrosine in HDL protein under
physiologically relevant in vitro conditions. To determine whether
myeloperoxidase can nitrate tyrosine residues on HDL protein, we
incubated the lipoprotein with the enzyme at neutral pH in phosphate
buffer containing NO2- (500 μM) and H2O2 (250
μM). We monitored the formation of 3-nitrotyrosine spectroscopically
by quantifying absorbance of the alkalinized reaction mixture at 430 nm.

[0112]3-Nitrotyrosine was readily detected in HDL exposed to the complete
myeloperoxidase-H2O2--NO2- system. Nitration required
each component of the reaction mixture: NO2-, H2O2,
and myeloperoxidase (FIG. 1A). The reaction depended on NO2concentration over a range of 0-1000 μM (FIG. 1B) and was complete in
20 min (FIG. 1C). It was inhibited by the peroxide scavenger catalase
(200 nM) (FIG. 1C) and the heme poison sodium azide (10 mM) (data not
shown). These results indicate that myeloperoxidase nitrates HDL by a
reaction that requires active enzyme, NO2-, and H2O2.

[0113]Myeloperoxidase generates 3-nitrotyrosine by directly oxidizing
NO2-. It has been proposed that myeloperoxidase uses at least
two distinct pathways to generate reactive nitrogen species (Eiserich et
al., (1996) Journal of Biological Chemistry 271, 19199-19208). In the
first pathway, the enzyme uses H2O2 and Cl- to generate
HOCl, which then reacts with NO2- to form nitryl chloride, a
nitrating species. In the second pathway, myeloperoxidase uses a
one-electron reaction to directly oxidize NO2- to nitrogen
dioxide radical, NO2.sup.•. The radical might then oxidize
tyrosine directly or might react with the tyrosyl radical that
myeloperoxidase also generates (38,53).

[0114]To distinguish between these two pathways, we examined the effect of
plasma concentrations of chloride ion (Cl-) on nitration of HDL by
the myeloperoxidase-H2O2--NO2- system (FIG. 2). We
also determined whether taurine (2-aminoethanesulfonic acid), a potent
scavenger of HOCl, inhibited nitration by the myeloperoxidase or
HOCl--NO2-. The extent of HDL nitration by myeloperoxidase was
independent of Cl- (FIG. 2A). Taurine also had no effect when
myeloperoxidase nitrated HDL in the presence of Cl- (FIG. 2B).
Moreover, we were unable to detect 3-nitrotyrosine in HDL exposed to
HOCl--NO2- (FIG. 2C). These observations indicate that HOCl
produced by myeloperoxidase is not a major contributor to the nitration
of HDL.

[0115]Instead, the pathway likely involves direct oxidation of
NO2- by compound I (a complex of myeloperoxidase and
H2O2) and the reaction of the resulting NO2.sup.•
with tyrosyl radical (van Dalen et al., (2000) J Biol Chem 275,
11638-11644). It is noteworthy that myeloperoxidase preferentially
oxidizes NO2- under these conditions, despite the presence of
200-fold greater levels of Cl-.

[0118]Myeloperoxidase immunoreactivity was very prominent in intimal
mononuclear cells. We detected such positive cells in all regions of
atheroma, though immunoreactivity was especially evident in the
subendothelial space, fibrous cap, and lipid core as well as near
microvessels. We also detected extracellular myeloperoxidase
immunoreactivity, both around macrophages (FIG. 3D) and in the lipid core
of advanced atheromatous plaques (data not shown).

[0119]To establish which cells express myeloperoxidase, we immunostained
atherosclerotic tissue with antibodies to myeloperoxidase and HAM-56, a
specific marker for macrophages. Most myeloperoxidase-positive cells
reacted with HAM-56, indicating that they were macrophages (FIG. 3C).

[0120]Advanced plaques contained many cells that were positive for both
myeloperoxidase and HAM-56, though some HAM-56-positive macrophages were
negative for myeloperoxidase. These results indicate that human
atherosclerotic lesions contain a major population of macrophages that
express myeloperoxidase.

[0121]To determine whether reactive intermediates from myeloperoxidase
might nitrate intimal proteins, we compared patterns of immunostaining
for 3-nitrotyrosine and myeloperoxidase. These patterns were virtually
identical (FIG. 3B,D). Antibodies to both 3-nitrotyrosine and the enzyme
reacted with material that associated closely with macrophages or was in
the macrophages themselves. These observations raise the possibility that
apo A-I is targeted for nitration in atherosclerotic intima. They also
support the proposal that myeloperoxidase is an important pathway for
generating 3-nitrotyrosine in the human artery wall.

[0122]HDL isolated from human atherosclerotic lesions contains
3-nitrotyrosine. To determine whether reactive nitrogen species damage
lipoproteins in vivo, we quantified 3-nitrotyrosine in lesion HDL. We
isolated the HDL by sequential ultracentrifugation from atherosclerotic
tissue that was freshly harvested from patients undergoing carotid
endarterectomy. To prevent artifactual oxidation of lipoproteins, we used
buffers containing high concentrations of DTPA (a metal chelator) and BHT
(a lipid soluble antioxidant). Western blotting with a monospecific
rabbit antibody confirmed that lesion HDL contained a high concentration
of apo A-I and a range of apparently larger immunoreactive proteins (FIG.
4A). Quantitative Western blotting demonstrated that apo A-I accounted
for >50% of the protein in the HDL.

[0123]To quantify 3-nitrotyrosine, isolated HDL was delipidated,
hydrolyzed, and the amino acids in the hydrolysate isolated by
solid-phase extraction on a C18 column. The reisolated amino acids were
derivatized and analyzed by GC/MS with selected ion monitoring in the
negative-ion chemical ionization mode. The derivatized amino acids
isolated from lesion HDL contained a compound that exhibited the major
ion identical to that of authentic 3-nitrotyrosine. Selected ion
monitoring showed that this ion (FIG. 4B) co-eluted with the ion derived
from 13C-labeled internal standard
(3-nitro[13C6]tyrosine). In contrast, there was little evidence
for 3-nitrotyrosine formation during sample work-up and analysis
(3-nitro[13C9,15N]tyrosine; FIG. 4B). These results
indicate that 3-nitrotyrosine is present in HDL isolated from human
atherosclerotic lesions and that it is not an artifact of sample
preparation.

[0124]HDL isolated from human atherosclerotic lesions is enriched in
3-nitrotyrosine. To assess quantitatively the contribution of nitration
to the oxidation of artery wall lipoproteins, we isolated HDL from plasma
of healthy humans and from human atherosclerotic aortic tissue. HDL was
delipidated and hydrolyzed, the resulting amino acids were isolated and
derivatized, and the derivatized amino acids were quantified with isotope
dilution GC/MS with selected ion monitoring (FIG. 5A). The concentration
of 3-nitrotyrosine in HDL isolated from the atherosclerotic lesions was 5
times higher (619±178 μmol/mol Tyr; n=10) than that in circulating
HDL (118±39 μmol/mol Tyr; n=13; P<0.01). These observations
provide strong evidence that HDL is one target for damage by reactive
nitrogen intermediates in the human artery wall.

[0125]HDL modified by reactive nitrogen species circulates in the blood of
humans with established coronary artery disease. To determine whether
nitrated HDL also circulates in blood, we used isotope dilution GC/MS to
quantify 3-nitrotyrosine levels in HDL isolated by sequential
ultracentrifugation from the blood of healthy humans and humans with
established atherosclerosis. The subjects with atherosclerosis had
lesions documented by clinical symptoms and coronary angiography. The
healthy subjects were normolipidemic with no known history of coronary
artery disease.

[0127]Levels of 3-nitrotyrosine correlate strongly with those of
3-chlorotyrosine in circulating HDL but not lesion HDL. To determine
whether myeloperoxidase might promote protein nitration in vivo, we
assessed the relationship between 3-chlorotyrosine, a marker of protein
oxidation that is generated only by myeloperoxidase at plasma
concentrations of halide ion, and levels of 3-nitrotyrosine in both
circulating and lesion HDL (FIG. 6).

[0128]Linear regression analysis demonstrated a strong correlation between
levels of 3-chlorotyrosine and levels of 3-nitrotyrosine (r2=0.65;
P<0.01) in plasma HDL. In contrast, there was no significant
correlation (r2=0.18; P=0.15) between levels of 3-chlorotyrosine and
those of 3-nitrotyrosine in lesion HDL. These observations strongly
support the hypothesis that myeloperoxidase promotes the formation of
3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but suggest that
other pathways also produce 3-nitrotyrosine in atherosclerotic tissue.

[0135]All samples were manually injected using an on column injector. The
level of chlorotyrosine was quantified using the ratio between the ion of
m/z 489 derived from 3-chlorotyrosine
([M-Cl-t-butyl-dimethylsilyl]-) and the ion of m/z 495 derived from
3-chloro[13C6]chlorotyrosine. Potential artifact formation was
monitored as the appearance of ions at m/z 499 derived from
L-[13C9,15N]tyrosine added prior to sample work up. Under
our experimental conditions, artifact formation was <5% of total
3-chlorotyrosine. To quantify L-tyrosine, which is present at 10,000-fold
higher levels than the oxidation products, the sample was diluted 1:100
and analyzed in a separate injection. L-Tyrosine and
L-[13C6]tyrosine were quantified using the ions
([M-COO-t-butyl-dimethylsilyl]-) at m/z 407 and m/z 413,
respectively.

[0136]Two-Dimensional Liquid Chromatograph--Tandem MS Analysis.
LC-ESI-MS/MS analyses were performed in the positive ion mode with a
Finnigan Mat LCQ ProteomeX ion trap instrument (San Jose, Calif.) coupled
to a Surveyor (Finnigan, San Jose, Calif.) quaternary HPLC pump, which in
turn was interfaced with a strong cation exchange resin and a
reverse-phase column (McDonald, W. H., and Yates, J. R., 3rd. 2002. Dis
Markers 18:99-105.). A fully automated 8-cycle chromatographic run was
carried out on each sample. The SEQUEST algorithm was used to interpret
MS/MS spectra. Matches were visually assessed if unique peptides had
highly significant SEQUEST scores (Id.).

[0137]Cell Culture and Cholesterol Efflux. Baby hamster kidney (BHK) cells
expressing mifepristone-inducible human ABCA1 were generated as
previously described (35). Cellular cholesterol was labeled by adding 1
μCi/mL [3H]cholesterol (NEN Life Science Products) to the growth
medium. Twenty four hours later, strong expression of ABCA1 was induced
by incubating the cells for 20 h with DMEM containing 1 mg/mL bovine
serum albumin (DMEM/BSA) and 1 nM mifepristone (Vaughan et al., 2003. J
Lipid Res 44:1373-1380). To measure cholesterol efflux, mock- or
ABCA1-transfected cells were incubated with DMEM/BSA without or with HDL,
apo A-I, or peptide. After 2 to 4 h, the medium and cells were assayed
for [3H]cholesterol as described (Id.). Cholesterol efflux mediated
by HDL, apo A-I, or peptide was calculated as the percentage of total
[3H]cholesterol (medium plus cell) released into the medium after
subtracting the value obtained with DMEM/BSA alone.

[0138]Statistical analysis. Results represent means±SD. Differences
between two groups were compared using an unpaired Student's t-test.
Multiple comparisons were performed using one-way analysis of variance
(ANOVA; Graph Pad software, San Diego, Calif.). A P value<0.05 was
considered significant.

[0140]It was demonstrated previously that myeloperoxidase is present in
atherosclerotic lesions, in both macrophage-associated and extracellular
distributions (Daugherty et al., 1994. J Clin Invest 94:437-444.). The
vast majority of cell-associated myeloperoxidase immunoreactivity was
present in macrophages, and most of the extracellular myeloperoxidase was
juxtaposed with macrophages (FIG. 7C,D). HOCl-modified proteins also
co-localized with macrophages. However, the most robust staining for
HOCl-modified proteins was extracellular and co-localized with apo A-I.
These observations are consistent with HOCl's ability to generate
long-lived reactive intermediates such as chloramines, which can diffuse
long distances to react with proteins. Indeed, chloramines mediate
tyrosine chlorination in apo A-I in vitro (Bergt et al., 2004. J Biol
Chem 279:7856-7866.). The co-localization of HOCl-modified proteins with
apo A-I suggests that HOCl oxidizes specific proteins in the human artery
wall.

[0142]We used negative-ion chemical ionization GC/MS to determine whether
3-chlorotyrosine was present in HDL isolated from human atherosclerotic
lesions. To confirm that any 3-chlorotyrosine detected in HDL was
endogenous rather than artifactual, an isotope-labeled tyrosine
(L-[13C9, 15N]tyrosine) was routinely added to each sample
before analysis. We reasoned that any procedure that converted endogenous
tyrosine to 3-chlorotyrosine would also convert
L-[13C9,15N]tyrosine to
3-chloro[13C9,15N]tyrosine. The latter would be detectable
by GC/MS because its mass-to-charge ratio (m/z) differs from those of
3-chlorotyrosine and the internal standard.

[0143]A compound was detected in the amino acid hydrolysate that exhibited
major ions and retention times identical to those of authentic
3-chlorotyrosine. Selected ion monitoring showed that the ions derived
from this amino acid co-eluted with those derived from
3-chloro[13C6]tyrosine (FIG. 2B). In contrast, there was little
evidence for 3-chlorotyrosine formation during sample work-up and
analysis (3-chloro[13C9, 15N]tyrosine).

[0145]HDL was isolated from human plasma and from human atherosclerotic
aortic tissue. After delipidating and hydrolyzing the proteins, levels of
the derivatized amino acid in acid hydrolysates were quantified with
isotope dilution GC/MS (FIG. 9A). Remarkably, there was six fold higher
level of protein-bound 3-chlorotyrosine in lesion HDL (177±27
μmol/mol Tyr; n=10) than in circulating HDL (28±7 μmol/mol Tyr;
n=13) isolated from humans (P<0.001).

[0146]HDL Isolated from Human Atherosclerotic Lesions Contains
Myeloperoxidase. Previous studies have shown that LDL binds
myeloperoxidase under physiologically relevant conditions (Carr et al.,
FEBS Lett 487:176-180). To determine whether HDL in the artery wall might
behave similarly, we digested lesion HDL with trypsin and analyzed the
resulting peptides with 2-D liquid chromatography and ESI-MS. Four
peptides in the digest were derived from myeloperoxidase. Their origin
was confirmed by sequencing them with MS/MS (FIG. 10). This observation
provides strong evidence that myeloperoxidase is a component of HDL
isolated by ultracentrifugation from atherosclerotic lesions and suggests
that the enzyme has high affinity for HDL in the artery wall.

[0147]Levels of 3-Chlorotyrosine Are Elevated in Plasma HDL from Humans
with Coronary Artery Disease. To determine whether oxidized HDL might
also be present in the circulation, we isolated HDL from plasma of
healthy subjects (4 males, ages 34-63) and subjects with established
coronary artery disease (7 males and 2 females, ages 33-67). The former
had no known history of vascular disease or symptoms suggestive of
angina, peripheral vascular disease, or cerebral vascular disease. The
subjects with coronary artery disease had angiographically documented
atherosclerosis.

[0148]To determine whether levels of chlorinated lipoproteins were
elevated in the subjects with coronary artery disease, we isolated HDL
from their plasma and plasma of healthy subjects. After delipidating and
hydrolyzing the proteins, we subjected the derivatized amino acid
hydrolysate to isotope dilution GC/MS analysis (FIG. 9B). The level of
protein-bound 3-chlorotyrosine was 8-times higher in circulating HDL from
the patients (39±7 μmol/mol Tyr; n=9) than in circulating HDL from
the healthy subjects (5±4 μmol/mol Tyr; n=4; P=0.01). Levels of
chlorinated HDL (perhaps derived from vascular lesions) are elevated in
the blood of humans suffering from clinically significant
atherosclerosis.

[0149]Oxidation of HDL and Apo A-I Impairs Cholesterol Transport in
Cultured Cells by ABCA1. The 10 amphipathic helices in apolipoprotein
A-I, HDL's major protein, are thought to play essential roles in lipid
binding, lipoprotein stability, and reverse cholesterol transport
(Segrest et al., 1992. J Lipid Res 33:141-166; Brouillette et al., 2001.
Biochim Biophys Acta 1531:4-46.). Five of the 7 tyrosine residues in this
protein lie in amphipathic helices, and we have previously shown that
Tyr192 in helix 8 is the major site of chlorination (Bergt et al., 2004.
J Biol Chem 279:7856-7866.). We therefore hypothesized that HOCl might
alter the ability of HDL and apo A-I to remove cholesterol from cells.

[0150]We exposed HDL or purified apo A-I to HOCl or H2O2 (80:1
or 25:1, mol/mol, oxidant:HDL particle or oxidant:apo A-I) in a
physiological buffer (138 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate) at
neutral pH for 120 min at 37° C., terminating the reaction with a
20-fold molar excess (relative to oxidant) of methionine. Because the
average HDL3 particle contains 2 mol of apolipoprotein A-I (7
tyrosine residues, 243 amino acids) and 1 mol of apolipoprotein A-II (8
tyrosine residues, 154 amino acids), the ratio of oxidant to substrate
(mol:mol) was ˜30:1 for apolipoproteins A-I and A-II, 3:1 for
tyrosine residues, and 1:8 for total amino acids. For lipid-free apo A-I,
the ratio of oxidant to substrates was ˜30% greater than for apo
A-I in HDL. We previously showed that ˜50% of Tyr192 is chlorinated
by HOCl under these conditions (Bergt et al., 2004. J Biol Chem
279:7856-7866).

[0151]We next determined how oxidation affects the ability of HDL or apo
A-I to promote cholesterol efflux from BHK cells that expressed very low
or very high levels of ABCA1. With mock-transfected cells (low ABCA1),
HDL promoted cholesterol efflux exclusively by diffusional mechanisms,
and apo A-I had essentially no cholesterol efflux activity (FIG. 5A).
Oxidation of HDL with HOCl or H2O2 (which oxidizes methionines)
had no effect on or slightly increased HDL-mediated cholesterol efflux
from these cells. When ABCA1 was overexpressed in transfected BHK cells,
however, HDL-mediated cholesterol efflux increased and apo A-I became
active (FIG. 11A). Whereas H2O2 oxidation had no effect,
chlorination was associated with a significant decrease in the
cholesterol efflux that was promoted by HDL or apo A-I (FIG. 11A, B).
These observations indicate that oxidation of HDL and apo A-I with HOCl
selectively impairs their abilities to remove cholesterol from cells by a
pathway requiring ABCA1.

[0153]We investigated the ability of native and oxidized 18A to promote
cholesterol efflux from BHK cells. In contrast to apo A-I, 18A promoted
cholesterol efflux from both mock- and ABCA1-transfected BHK cells, but
to a much greater extent from the ABCA1-expressing cells. HOCl treatment
significantly reduced 18A's ability to remove cholesterol by both the
ABCA1-independent and -dependent mechanisms (FIG. 11C). Site-specific
oxidation of tyrosines in amphipathic a-helices can impair lipid
transport activities.

Example 3

[0154]Approximately 40% of renal transplants are performed in diabetics.
These patients are at high risk for atherosclerosis and approximately 50%
of the transplants are lost due to cardiovascular mortality in these
patients despite acceptable renal graft function. Kidney disease has been
linked to risk of recurrent cardiovascular disease and mortality. See,
Shlipak et al, NEJM, (2004) 352(20):2049; Coresh et al., (2005)
Circulation and Hemodynamics 10:73; Weiner et al., American Journal of
Kidney Diseases (2004) 44(2):198; Anavekar et al., (2004) NEJM
351(13):1285; Go et al., NEJM (2004) 351(13):1296. We postulated that
oxidative stress is increased in the diabetic renal transplant patient
population.

Methods

[0155]We divided a study population into 2 groups according to
non-diabetic control patients who have undergone a renal transplant and
diabetic patients who have undergone a renal transplant. Ten patients
were included in each group. Serum creatinine levels were measured for
patients in each group to verify that there was no significant difference
between the two groups (mean 1.7 vs 1.62 mg/dL). The mean HbA1C for the
diabetic patients was 8.3. End stage renal disease (ESRD) secondary to
diabetes was a prerequisite to be enrolled in the diabetic arm. All
patients were required to have stable renal function for at least 3
months after renal transplant, creatinine<1.8 mg/dl (estimated
creatinine clearance by CG formula of >50 ml/min), proteinuria<250
mg/day based on average of three measurements of spot protein/creatinine
ratio, no active infection or evidence of rejection and no clinically
active coronary artery disease (CAD).

[0157]Two-Dimensional Liquid Chromatography--Tandem MS Analysis.
LC-ESI-MS/MS analyses were performed in the positive ion mode with a
Finnigan Mat LCQ ProteomeX ion trap instrument (San Jose, Calif.) coupled
to a Surveyor (Finnigan, San Jose, Calif.) quaternary HPLC pump, which in
turn was interfaced with a strong cation exchange resin and a
reverse-phase column (McDonald, W. H., and Yates, J. R., 3rd. 2002. Dis
Markers 18:99-105). A fully automated 8-cycle chromatographic run was
can-fed out on each sample. The SEQUEST algorithm was used to
interpret MS/MS spectra. Matches were visually assessed if unique
peptides had highly significant SEQUEST scores (Id.).

[0158]Statistical analysis. Results represent means±SD. Differences
between the two patient populations were compared using an unpaired
Student's t-test. Multiple comparisons were performed using one-way
analysis of variance (ANOVA; Graph Pad software, San Diego, Calif.). A P
value<0.05 was considered significant.

[0159]HDL was isolated from urine, human plasma and from human
atherosclerotic aortic tissue in patients having undergone renal
transplant and in control patients. After delipidating and hydrolyzing
the proteins, levels of the derivatized amino acid in acid hydrolysates
were quantified with isotope dilution GC/MS pursuant to the protocols
outlined in Example 2.

[0160]Dityrosine levels present in the urine of diabetic patients having
undergone a renal transplant are elevated in comparison to control
patients as depicted in FIG. 12. Levels of circulating nitrotyrosine
present in the plasma in diabetic patients having undergone a renal
transplant are also elevated relative to control patients as depicted in
FIG. 13. Similarly, myeloperoxidase levels present in the plasma in
diabetic patients having undergone a renal transplant are elevated in
comparison to control patients.

Example 4

[0161]HDL isolated from carotid atherosclerotic tissue in diabetic
patients contains 3-nitrotyrosine and 3-chlorotyrosine in amounts greater
than found in HDL isolated from the plasma of control patients. We
quantified 3-nitrotyrosine and 3-chlorotyrosine in HDL isolated from
atherosclerotic tissue obtained from diabetic patients and from the
plasma of non-diabetic patient groups described in Example 3. The
quantification was performed according to the methods set forth in
Example 1. We isolated the HDL by sequential ultracentrifugation from
atherosclerotic tissue that was freshly harvested from patients. To
prevent artifactual oxidation of lipoproteins, we used buffers containing
high concentrations of DTPA (a metal chelator) and BHT (a lipid soluble
antioxidant). Western blotting with a monospecific rabbit antibody
confirmed that lesion HDL contained a high concentration of apo A-I and a
range of apparently larger immunoreactive proteins. Quantitative Western
blotting demonstrated that apo A-I accounted for >50% of the protein
in the HDL.

[0162]To quantify 3-nitrotyrosine and 3-chlorotyrosine, isolated HDL was
delipidated, hydrolyzed, and the amino acids in the hydrolysate isolated
by solid-phase extraction on a C18 column. The reisolated amino acids
were derivatized and analyzed by GC/MS with selected ion monitoring in
the negative-ion chemical ionization mode. The derivatized amino acids
isolated from the HDL obtained from carotid atherosclerotic tissue
contained compounds that exhibited the major ions identical to that of
3-nitrotyrosine and 3-chlorotyrosine. Selected ion monitoring showed that
these ions co-eluted with the ion derived from 13C-labeled internal
standard.

[0163]To assess quantitatively the contribution of nitration to the
oxidation of artery wall lipoproteins, we isolated HDL from plasma of the
control patients. HDL was delipidated and hydrolyzed, the resulting amino
acids were isolated and derivatized, and the derivatized amino acids were
quantified with isotope dilution GC/MS with selected ion monitoring. The
concentration of 3-nitrotyrosine in HDL isolated from the atherosclerotic
lesions of the diabetic patients was higher than that in HDL of the
normal patients as depicted in FIG. 20.